PROJECT SUMMARY Proline isomerization is emerging as a significant mechanism of regulating cell signaling, transcription and the cell cycle. Determination of the proline isomerization state in vivo is extremely challenging with existing methods. We propose to incorporate unique molecular tools, cis-locked and trans-locked Pro analogues, into full-length proteins to permit analysis of conformation-specific activity in living cells (Figure 1). We will insert our locked alkene analogues into Protein Aurora Borealis (Bora) using in vitro transcription-translation as a proof-of-principle for the method. We will validate locked protein tools against a known outcome, activation of Aurora A by Bora in living cells. The effect of proline isomerization states of Bora, with and without phosphate, will be investigated in vitro, and phenotypically in living cells. The overall goal of this work is to develop a robust method to insert locked cis- and trans-Pro analogues into full-length proteins. Developing the tools to examine the effects of proline isomerization state in living cells is critical to a broader understanding of cell signaling, transcription, and cell cycle regulation. These tools will enable determination of the precise isomer of cis- or trans-Pro that is active in a specific cellular process. To achieve these goals, we will pursue the following aims. Aim 1. Incorporate cis- and trans-locked Ser-Pro analogues into full-length proteins. Native and conformationally locked full-length Bora proteins will be synthesized in an E. coli expression system with tRNA chemically misacylated with locked Ser-Pro dipeptide analogues. Proteins will be verified as substrates for the proline-directed Ser/Thr protein kinases, GSK3 and Cdk1-cyclin B in vitro. The phosphorylation state at the Ser- Pro analogue positions will be determined by LC-MSMS. Aim 2. Incorporate phosphorylated cis- and trans-locked Ser-Pro analogues into full-length proteins. Native and conformationally locked phosphorylated Bora proteins will be synthesized in an E. coli expression system with tRNA chemically misacylated with phosphorylated and locked Ser-Pro dipeptide analogues. The effect of locked phosphorylated Bora proteins on Aurora A kinase activity will be verified in vitro. Aim 3. Demonstrate the feasibility of using locked full-length proteins as a tool in cell biology. Full-length Bora proteins with locked Ser-Pro dipeptide analogues, in both unphosphorylated and phosphorylated states, will be microinjected into cell cycle synchronized human cells depleted of the endogenous Bora protein, and the cell phenotypes will be evaluated by live-cell microscopy.